It has been known for decades that viruses induce dramatic changes to host-cell metabolism and that these changes are important for viral replication. The mechanisms involved, however, have largely remained obscure. A major challenge in dissecting the mechanisms of viral metabolic manipulation has been the technical difficulty associated with measuring diverse metabolic activities in live cells. We have developed a liquid chromatography-tandem mass spectrometry-based (LC-MS/MS) methodology to measure global metabolic activities in live cells. We propose that elucidation of these viral mechanisms will prove fertile ground for the development of novel anti-viral therapeutics. Our results indicate that three metabolic regulatory activities are required for high-titer HCMV replication; AMP-activated kinase (AMPK) and phosphofructokinase-1 (PFK1), both of which regulate glycolytic flux, and acetyl-CoA carboxylase (ACC1), which regulates fatty acid biosynthesis. In uninfected cells, activated AMPK directly inhibits ACC1 activity and thereby, fatty acid biosynthesis. We find that HCMV blocks this regulatory control, maintaining increased ACC1 activity despite activated AMPK. This results in a dramatic activation of both glycolysis and fatty acid biosynthesis. To explore these mechanisms, we will: (I) Elucidate the mechanisms of HCMV-induced AMPK activation and its role in HCMV replication; (II) Elucidate the mechanisms of HCMV- induced ACC1 activation; and (III) Elucidate the mechanisms of HCMV-induced PFK1 activation and how they contribute to viral replication. Through elucidating the viral mechanisms leading to the activation of these metabolic activities we will identify novel anti-viral targets to combat HCMV-associated disease and further explore viral manipulation of these fundamental host-cell pathways. PUBLIC HEALTH RELEVANCE: Human Cytomegalovirus (HCMV) is a widespread opportunistic pathogen that can cause severe disease in various immunosuppressed populations including the elderly, cancer patients receiving immunosuppressive chemotherapy, transplant recipients, and AIDS patients. HCMV is also the leading cause of congenital viral infection, occurring in 1-2% of all live births, which can result in multiple organ system abnormalities with central nervous system damage occurring in the majority of symptomatic newborns. Long term use of current anti-HCMV therapeutics in patients leads to toxic side effects and has resulted in the emergence of drug-resistant viral strains, highlighting the need for additional anti-HCMV therapeutics. Our proposed research aims to elucidate the mechanisms HCMV utilizes to drive the biosynthesis of virion components. Elucidating these mechanisms will present targets to therapeutically block viral replication and attenuate HCMV-associated disease.